The virus population bottleneck to Hepatitis C Virus (HCV) and Human Immunodeficiency Virus type 1 (HIV-1) transmission in injection drug users (IDUs) remains an understudied and controversial area of research with substantial potential relevance to prevention efforts, including vaccine design and assessment. Single genome sequencing (SGS) represents a powerful strategy for identifying and characterizing viruses that are responsible for establishing productive clinical infection (termed transmitted/founder, or T/F, viruses). Scientists have only recently begun to apply this approach to HIV and HCV transmission in IDUs; there are just two small studies enumerating HIV-1 T/F viruses in IDU subjects with conflicting conclusions, and a single study of HCV transmission in plasma donors where IDU were technically excluded. Importantly, these HIV-1 and HCV cohorts had neither longitudinal sampling nor comprehensive behavioral data available, which are critical in IDUs to distinguish between sexual and injection-related virus acquisition. In this application, we propose to combine novel quantitative and qualitative analyses of the HIV-1 and HCV T/F viruses responsible for productive clinical infection with a detailed assessment of injection and sexual transmission risk behaviors in a robust and clinically well-defined study population of 52 HIV-1 seroconversions and 71 HCV seroconversions from two recent HIV Prevention Trials Unit studies in Xinjiang, China. This application tests the hypothesis that IDU transmission is governed by quantifiable behavioral risks that determine the rates of virus acquisition, the multiplicity of virus infection, the virus phenotype and clinical outcomes of HIV and HCV infection. We will address this hypothesis by (i) identifying and enumerating the T/F HIV-1 and HCV viruses from the Xinjiang IDUs with acute HIV-1 and HCV infection, (ii) inferring the mode of transmission (sexual vs. injection) and determining the behavioral correlates of HIV-1 and HCV acquisition and multiple virus transmission, (iii) assessing the role of multiplicity of virus infection on HIV and HCV clinical outcomes, and (iv) molecularly cloning and determining the biologic phenotype of T/F HIV-1 Envs and the replication competence of HCV full-length genomes from this cohort of Chinese IDUs. Accurate characterization of the HIV and HCV transmission bottleneck in IDUs and its role in clinical disease progression will provide key information relevant to HIV-1 and HCV transmission, natural history and prevention, and in addition, will provide essential virologic data and molecular reagents for vaccine research focused on HIV-1 and HCV strains relevant to China, Southeast and Central Asia.